Helicobacter pylori infects approximately half of the world's population and is responsible for inducing chronic gastric inflammation that can progress to gastric cancer (1). At the cellular level, Helicobacter pylori infection of the human stomach is associated with inflammation that elicits DNA damage in both bacterial and host cells (2). This DNA damage must be repaired in order for the bacteria to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization (3), and inhibitors of this enzyme may be useful antibacterial drugs for treating these infections. The AddAB class of enzymes is closely related to the RecBCD class of helicase-nucleases; both classes are widely distributed in bacteria but appear to be absent in eukaryotes (4). The protein complex functions in DNA repair by directing free DNA ends into the homologous recombination pathway (5). As a result, the identification of inhibitors of AddAB may be useful tools for elucidating the role of AddAB and RecBCD in bacterial recombination and as potential novel antibiotics with few off-target effects.

Assay Overview:The purpose of this assay is to determine RecBCD inhibition dose response curves for powder samples of compounds identified as possible AddAB probe candidates. Compounds that also inhibit E. coli RecBCD may have potential to elicit a broad-spectrum antibiotic like effect. This assay involves infecting E. coli with a T4 bacteriophage that carries three nonsense mutations in gene 2, whose wild-type protein product protects viral DNA from RecBCD-mediated degradation after infection. The mutant T4 phage is able to infect and block the growth of V67 E. coli (recB21, a recBCD null mutation), which lack RecBCD nuclease activity. The mutant phage also infects V66 E. coli (recBCD+), but V66 proliferate because of the RecBCD helicase and nuclease activity against the unprotected mutant phage. In this assay, the V66 E.coli cells are infected with mutant T4 phage in the presence of test compounds, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit RecBCD will allow the virus to replicate and block bacterial growth, leading to reduced well absorbance. Compounds are tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 118.6 uM.Protocol Summary:Prior to the start of the assay, V66 and V67 bacterial cultures were grown at 37 C until it reached an OD600 of 0.05 or 2.5e07 cfu/mL. To start the assay, 3 uL of Assay Buffer (0.1% Glycerol + Cation Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 ug/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells. Then, 1 uL of V66 (recBCD+) or V67 (phage control) bacterial cultures were dispensed into the appropriate wells and plates were incubated for 60 minutes at 37 C.Next, 1 uL of mutant T4 2 149 bacteriophage was dispensed to the appropriate wells at a multiplicity of infection (MOI) of 0.02. Plates were centrifuged and after 18 hours of incubation at 37 C, absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well.The percent inhibition for each compound was calculated as follows:%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )Where:High_Control is defined as wells containing V66 + Ciprofloxacin + phageLow_Control is defined as wells containing V66 + DMSO + phage.Test_Compound is defined as wells containing V66 in the presence of test compound + phage.For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 118.6 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 118.6 uM.PubChem Activity Outcome and Score:Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.List of Reagents:V66 (recBCD+) and V67 (recB21) E. coli bacteria (supplied by Assay Provider)T4 2 149 mutant bacteriophage (supplied by Assay Provider)Ciprofloxacin (Sigma, part 17850)Cation-Adjusted Mueller Hinton II Broth (BD, part 297963)1536-well plates (Aurora, part 19326)

This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.

Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.

String

2

IC50*

The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.

Float

μM

3

LogIC50

Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration

Float

4

Maximal Response

The maximal or asymptotic response above the baseline as concentration increases without bound.

Float

5

Baseline Response

Adjustable baseline of the curve fit, minimal response value.

Float

6

Inflection Point Concentration

The concentration value for the inflection point of the curve.

Float

μM

7

Hill Slope

The variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.

Float

8

Hill S0

Y-min of the curve.

Float

9

Hill Sinf

Y-max of the curve.

Float

10

Hill dS

The range of Y.

Float

11

Chi Square

A measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.

Float

12

Rsquare

This statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.

Float

13

Number of DataPoints

Overall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.